It is well established that Alzheimer's disease and a number of related tauopathies including Downs' syndrome, Pick's disease, Niemann-Pick Type C disease, and Amyotrophic lateral sclerosis are characterized, in part, by the development of neurofibrillary tangles (NFTs). These NFTs are aggregates of paired helical filaments (PHFs) and are composed of an abnormal form of the cytoskeletal protein “tau”. Normally tau stabilizes a key cellular network of microtubules that is essential for distributing proteins and nutrients within neurons. In Alzheimer's disease patients, however, tau becomes hyperphosphorylated, disrupting its normal functions, forming PHFs and ultimately aggregating to form NFTs. Six isoforms of tau are found in the human brain. In Alzheimer's disease patients, all six isoforms of tau are found in NFTs, and all are markedly hyperphosphorylated (Goedert et al., Neuron 1992, 8, 159; and Goedert et al., Neuron 1989, 3, 519).
Tau in healthy brain tissue bears only 2 or 3 phosphate groups, whereas those found in the brains of Alzheimer's disease subjects bear, on average, 8 phosphate groups (Kopke et al., J Biol Chem 1993, 268, 24374; and Ksiezak-Reding et al., Brain Res 1992, 597, 209). A clear parallel between NFT levels in the brains of Alzheimer's disease patients and the severity of dementia strongly supports a key role for tau dysfunction in Alzheimer's disease (Arriagada et al., Neurology 1992, 42, 631; Riley et al., Ann Neurol 2002, 51, 567; and Alafuzoff et al., Acta Neuropathol (Berl) 1987, 74, 209). Accordingly, approaches aimed at reducing NFTs and/or hyperphosphorylated tau represent potential disease modifying treatments for Alzheimer's disease.
It is also well-established that a wide range of cellular proteins, both nuclear and cytoplasmic, are post-translationally modified by the addition of the monosaccharide 2-acetamido-2-deoxy-β-D-glucopyranoside (β-N-acetylglucosamine) which is attached via an O-glycosidic linkage (Torres et al., J Biol Chem 1984, 259, 3308). This modification is generally referred to as O-linked N-acetylglucosamine or O-GlcNAc. The enzyme responsible for post-translationally linking β-N-acetylglucosamine (GlcNAc) to specific serine and threonine residues of numerous nucleocytoplasmic proteins is O-GlcNAc transferase (OGT) (Haltiwanger et al., J Biol Chem 1990, 265, 2563; Kreppel et al., J Biol Chem 1997, 272, 9308; Lubas et al., J Biol Chem 1997, 272, 9316; and Lubas et al., J Biol Chem 2000, 275, 10983). A second enzyme, known as O-linked N-acetylglucosamin-dase (O-GlcNAcase) (Dong et al., J Biol Chem 1994, 269, 19321; and Gao et al., J Biol Chem 2001, 276, 9838) removes this post-translational modification to liberate proteins making the O-GlcNAc-modification a dynamic cycle occurring several times during the lifetime of a protein (Roquemore et al., Biochemistry 1996, 35, 3578).
It has recently emerged that phosphate levels of tau are regulated, in part, by the levels of O-Glc-NAc on tau. The presence of O-GlcNAc on tau has stimulated studies that correlate O-GlcNAc levels with tau phosphorylation levels. In this regard, it has been found that increases in phosphorylation levels result in decreased O-GlcNAc levels and conversely, increased O-GlcNAc levels correlate with decreased phosphorylation levels (Griffith et al., Eur J Biochem 1999, 262, 824). Hyperphosphorylated tau in human Alzheimer's disease brains has markedly lower levels of O-GlcNAc than are found in healthy human brains (Liu et al., Proc Natl Acad Sci USA 2004, 101, 10804).
Very recently, it has been shown that O-GlcNAc levels of soluble tau protein from human brains affected with Alzheimer's disease are markedly lower than those from healthy brain (Liu et al., Brain, 2009, 132, 1820).
Recent studies (Yuzwa et al., Nat Chem Biol 2008, 4, 483) support the therapeutic potential of small-molecule O-GlcNAcase inhibitors to limit tau hyperphosphorylation for treatment of Alzheimer's disease and related tauopathies. Specifically, the O-GlcNAcase inhibitor thiamet-G has been implicated in the reduction of tau phosphorylation in cultured PC-12 cells at pathologically relevant sites and in the brains of animals after in vivo administration of this inhibitor (Yuzwa et al., supra). Accordingly, O-GlcNAcase inhibitors are widely recognized as a valid therapeutic approach to reduce hyperphosphorylation of tau and formation of NFTs.
There is also a large body of evidence indicating that increased levels of O-GlcNAc protein modification provides protection against pathogenic effects of stress in cardiac tissue, including stress caused by ischemia, hemorrhage, hypervolemic shock, and calcium paradox. For example, activation of the hexosamine biosynthetic pathway (HBP) by administration of glucosamine has been demonstrated to exert a protective effect in animals models of ischemia/reperfusion (Bounelis et al., Shock 2004, 21 170 Suppl. 2, 58; Fulop et al., Circulation Research 2005, 97, E28; Liu et al., Faseb Journal 2006, 20, A317; Marchase et al., PCT Int. App. WO 2006016904 2006; Fulop et al., Journal of Molecular and Cellular Cardiology 2004, 37, 286; Fulop et al., Faseb Journal 2005, 19, A689; and Liu et al., Journal of Molecular and Cellular Cardiology 2007, 42, 177), trauma hemorrhage (Not et al., Faseb Journal 2006, 20, A1471; Yang et al., Shock 2006, 25, 600; and Zou et al., Faseb Journal 2005, 19, A1224), hypervolemic shock (Marchase et al., Circulation 2004, 110, 1099) and calcium paradox (Bounelis et al., supra; and Liu et al., Journal of Molecular and Cellular Cardiology 2006, 40, 303). Moreover, strong evidence indicates that these cardioprotective effects are mediated by elevated levels of protein O-GlcNAc modification (Bounelis et al., supra; Fulop et al., Circulation Research 2005, 97, E28; Marchase et al., 2006, supra; Liu et al., 2007, supra; Yang et al., supra; Liu et al., Journal of Molecular and Cellular Cardiology 2006, 40, 303; Liu et al., Faseb Journal 2005, 19, A691; Nagy et al., American Journal of Physiology-Cell Physiology 2006, 290, C57; and Fulop et al., Cardiovascular Research 2007, 73, 288). There is also evidence that the O-GlcNAc modification plays a role in a variety of neurodegenerative diseases, including Parkinson's disease and Huntington's disease (Lefebvre et al., Expert Review of Proteomics 2005, 2, 265).
Humans have three genes encoding enzymes that cleave terminal β-N-acetylglucosamine residues from glycoconjugates. The first of these encodes the enzyme O-glycoprotein 2-acetamido-2-deoxy-β-D-glucopyranosidase (O-GlcNAcase) as is indicated above. O-GlcNAcase is a member of family 84 of glycoside hydrolases that includes enzymes from organisms as diverse as prokaryotic pathogens to humans (for the family classification of glycoside hydrolases see Coutinho, P. M. & Henrissat, B. (1999) Carbohydrate-Active Enzymes server at URL: http://afmb.cnrs-mrs.fr/CAZY/ (Henrissat et al., Biochem J 1996, 316 (PT2), 695; and Henrissat et al., 1993, supra). O-GlcNAcase acts to hydrolyse O-GlcNAc off of serine and threonine residues of post-translationally modified proteins (Torres et al., supra; Dong et al., supra; Gao et al., supra; Wells et al., Science 2001, 291, 2376; and Hanover, Faseb Journal 2001, 15, 1865). Consistent with the presence of O-GlcNAc on many intracellular proteins, the enzyme O-GlcNAcase appears to have a role in the etiology of several diseases including type II diabetes (Volleller et al., Proc Natl Acad Sci USA 2002, 99, 5313; and McClain et al., Proc Natl Acad Sci USA 2002, 99, 10695), A D (Griffith, Biochem Biophys Res Commun 1995, 213, 424; Liu et al., Proc Natl Acad Sci USA 2004, 101, 10804; and Yao et al., J. Neurosci 1998, 18, 2399) and cancer (Chou et al., Adv Exp Med Biol 2001, 491, 413; and Yang et al., Nature Cell Biology 2006, 8, 1054). Although O-GlcNAcase was likely isolated earlier on (Braidman et al., Biochem J 1974, 143, 295; and Ueno et al., Biochim Biophys Acta 1991, 1074, 79), about 20 years elapsed before its biochemical role in acting to cleave O-GlcNAc from serine and threonine residues of proteins was understood (Dong et al., supra). More recently O-GlcNAcase has been cloned (Gao et al., supra), partially characterized (Toleman et al., J Biol Chem 2004) and suggested to have additional activity as a histone acetyltransferase (Toleman et al., supra). However, little was known about the catalytic mechanism of this enzyme.
The other two genes, HEXA and HEXB, encode enzymes catalyzing the hydrolytic cleavage of terminal β-N-acetylglucosamine residues from glycoconjugates. The gene products of HEXA and HEXB predominantly yield two dimeric isozymes, hexosaminidase A and hexosaminidase B, respectively. Hexosaminidase A (αβ), a heterodimeric isozyme, is composed of an α- and a β-subunit. Hexosaminidase B (ββ), a homodimeric isozyme, is composed of two β-subunits. The two subunits, α- and β-, bear a high level of sequence identity. Both of these enzymes are classified as members of family 20 of glycoside hydrolases and are normally localized within lysosomes. The proper functioning of these lysosomal β-hexosaminidases is critical for human development, a fact that is underscored by the tragic genetic illnesses, Tay-Sach's and Sandhoff diseases which stem from a dysfunction in, respectively, hexosaminidase A and hexosaminidase B (Triggs-Raine et al., Adv Genet, 2001, 44, 199). These enzymatic deficiencies cause an accumulation of glycolipids and glycoconjugates in the lysosomes resulting in neurological impairment and deformation. The deleterious effects of accumulation of gangliosides at the organismal level are still being uncovered (Zhou et al., Science 2004).
As a result of the biological importance of these β-N-acetyl-glucosaminidases, small molecule inhibitors of glycosidases (Legler et al., Biochim Biophys Acta 1992, 1080, 89; Horsch et al., Eur J. Biochem 1991, 197, 815; Liu et al., Chem Biol 2001, 8, 701; and Knapp et al., J Am Chem Soc 1996, 118, 6804) have received a great deal of attention (Lillelund et al., Chem Rev 2002, 102, 515), both as tools for elucidating the role of these enzymes in biological processes and in developing potential therapeutic applications. The control of glycosidase function using small molecules offers several advantages over genetic knockout studies including the ability to rapidly vary doses or to entirely withdraw treatment.
However, a major challenge in developing inhibitors for blocking the function of mammalian glycosidases, including O-GlcNAcase, is the large number of functionally related enzymes present in tissues of higher eukaryotes. Accordingly, the use of non-selective inhibitors in studying the cellular and organismal physiological role of one particular enzyme is complicated because complex phenotypes arise from the concomitant inhibition of such functionally related enzymes. In the case of β-N-acetylglucosaminidases, existing compounds that act to block O-GlcNAcase function are non-specific and act potently to inhibit the lysosomal β-hexosaminidases.
A few of the better characterized inhibitors of β-N-acetyl-glucosaminidases which have been used in studies of O-GlcNAc post-translational modification within both cells and tissues are streptozotocin (STZ), 2′-methyl-α-D-glucopyrano-[2,1-d]-Δ2′-thiazoline (NAG-thiazoline) and O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate (PUGNAc) (Vosseller et al., supra; Konrad et al., Biochem J 2001, 356, 31; Liu et al., J Neurochem 2004, 89, 1044; Parker et al., J Biol Chem 2004, 279, 20636; and Arias et al., Diabetes 2004, 53, 921).
STZ has long been used as a diabetogenic compound because it has a particularly detrimental effect on β-islet cells (Junod et al., Proc Soc Exp Biol Med 1967, 126, 201). STZ exerts its cytotoxic effects through both the alkylation of cellular DNA (Junod et al., supra; and Bennett et al., Cancer Res 1981, 41, 2786) as well as the generation of radical species including nitric oxide (Kroncke et al., Biol Chem Hoppe Seyler 1995, 376, 179). The resulting DNA strand breakage promotes the activation of poly(ADP-ribose) polymerase (PARP) (Yamamoto et al., Nature 1981, 294, 284) with the net effect of depleting cellular NAD+ levels and, ultimately, leading to cell death (Yamada et al., Diabetes 1982, 31, 749; and Burkart et al., Nat Med 1999, 5, 314). Other investigators have proposed instead that STZ toxicity is a consequence of the irreversible inhibition of O-GlcNAcase, which is highly expressed within β-islet cells (Konrad et al., supra; and Roos, Proc Assoc Am Physicians 1998, 110, 422). This hypothesis has, however, been brought into question by two independent research groups (Gao et al., Arch Biochem Biophys 2000, 383, 296; and Okuyama et al., Biochem Biophys Res Commun 2001, 287, 366). Because cellular O-GlcNAc levels on proteins increase in response to many forms of cellular stress (Zachara et al., J Biol Chem 2004, 279, 30133) it seems possible that STZ results in increased O-GlcNAc-modification levels on proteins by inducing cellular stress rather than through any specific and direct action on O-GlcNAcase. Indeed, Hanover and coworkers have shown that STZ functions as a poor and somewhat selective inhibitor of O-GlcNAcase (Hanover et al., Arch Biochem Biophys 1999, 362, 38) and although it has been proposed by others that STZ acts to irreversibly inhibit O-GlcNAcase (Liu et al., Mol Cell Endocrinol 2002, 194, 135), there has been no clear demonstration of this mode of action. Recently, it has been shown that STZ does not irreversibly inhibit O-GlcNAcase (Macauley et al., J Biol Chem 2005, 280, 25313).
NAG-thiazoline has been found to be a potent inhibitor of family 20 hexosaminidases (Knapp et al., supra; and Mark et al., J Biol Chem 2001, 276, 10330), and more recently, the family 84 O-GlcNAcases (Macauley et al., supra). Despite its potency, a downside to using NAG-thiazoline in a complex biological context is that it lacks selectivity and therefore perturbs multiple cellular processes.
PUGNAc is another compound that suffers from the same problem of lack of selectivity, yet has enjoyed use as an inhibitor of both human O-GlcNAcase (Dong et al., supra; and Haltiwanger et al., J Biol Chem 1998, 273, 3611) and the family 20 human β-hexosaminidases (Miller et al., Development 1993, 118, 1279). This molecule, developed by Vasella and coworkers, was found to be a potent competitive inhibitor of the β-N-acetyl-glucosaminidases from Canavalia ensiformis, Mucor rouxii, and the β-hexosaminidase from bovine kidney (Horsch et al., supra). It has been demonstrated that administration of PUGNAc in a rat model of trauma hemorrhage decreases circulating levels of the pro-inflammatory cytokines TNF α and IL-6 (Zou et al., Shock 2007, 27, 402). It has also been shown that administration of PUGNAc in a cell-based model of lymphocyte activation decreases production of the cytokine IL-2 (Huang et al., Cellular Immunology 2007, 245, 1). Recent studies have indicated that PUGNAc can be used in an animal model to reduce myocardial infarct size after left coronary artery occlusion (U.J.G. Conference, in US/Japan Gylco 2004 Conference, Honolulu, Hi., 2004). Of particular significance is the fact that elevation of O-GlcNAc levels by administration of PUGNAc, an inhibitor of O-GlcNAcase, in a rat model of trauma hemorrhage improves cardiac function (Zou et al., Shock 2007, 27, 402; and Zou et al., Faseb Journal 2006, 20, A1471). In addition, elevation of O-GlcNAc levels by treatment with PUGNAc in a cellular model of ischemia/reperfusion injury using neonatal rat ventricular myocytes improved cell viability and reduced necrosis and apoptosis compared to untreated cells (Champattanachai et al., American Journal of Physiology-Cell Physiology 2007, 292, C178).
More recently, it has been suggested that the selective O-GlcNAcase inhibitor NButGT exhibits protective activity in cell-based models of ischemia/reperfusion and cellular stresses, including oxidative stress (Champattanachai et al., American Journal of Physiology-Cell Physiology 2008, 294, C1509). This study suggests the use of O-GlcNAcase inhibitors to elevate protein O-GlcNAc levels and thereby prevent the pathogenic effects of stress in cardiac tissue.
International patent applications PCT/CA2006/000300, filed 1 Mar. 2006, published under No. WO 2006/092049 on 8 Sep. 2006; PCT/CA2007/001554, filed 31 Aug. 2007, published under No. WO 2008/025170 on 6 Mar., 2008; PCT/CA2009/001087, filed 31 Jul. 2009, published under No. WO 2010/012106 on 4 Feb. 2010; PCT/CA2009/001088, filed 31 Jul. 2009, published under WO 2010/012107 on 4 Feb. 2010; and PCT/CA2009/001302, filed 16 Sep. 2009, published under WO 2010/037207 on 8 Apr. 2010, describe selective inhibitors of O-GlcNAcase.
Noninvasive nuclear imaging techniques can be used to obtain basic and diagnostic information about the physiology and biochemistry of a variety of living subjects including experimental animals, normal humans and patients. These techniques rely on the use of sophisticated imaging instrumentation that is capable of detecting radiation emitted from radiotracers administered to such living subjects. The information obtained can be reconstructed to provide planar and tomographic images that reveal distribution of the radiotracer as a function of time. Use of appropriately designed radiotracers can result in images which contain information on the structure, function and most importantly, the physiology and biochemistry of the subject. Much of this information cannot be obtained by other means. The radiotracers used in these studies are designed to have defined behaviors in vivo which permit the determination of specific information concerning the physiology or biochemistry of the subject or the effects that various diseases or drugs have on the physiology or biochemistry of the subject. Currently, radiotracers are available for obtaining useful information concerning such things as cardiac function, myocardial blood flow, lung perfusion, liver function, brain blood flow, brain regional distribution and function.
For noninvasive in vivo imaging, compounds can be labeled with either positron- or gamma-emitting radionuclides. The most commonly used positron emitting (PET) radionuclides are 11C, 18F, 15N and 13N, all of which are accelerator produced, and have half-lives of 20, 110, 2 and 10 minutes, respectively. These short half-lives endow a number of advantages to their use as tracers to probe biological processes in vivo using PET. Since the half-lives of these radionuclides are so short, it is only feasible to use them at institutions that have an accelerator on site or very close by for their production, thus limiting their use.
In a typical PET study, a small amount of radiotracer is administered to the experimental animal, normal human or patient being tested. The radiotracer then circulates in the blood of the subject and may be absorbed in certain tissues. The radiotracer may be preferentially retained in some of these tissues because of specific enzymatic conversion or by specific binding to macromolecular structures such as proteins. Using sophisticated imaging instrumentation to detect positron emission, the amount of radiotracer is then non-invasively assessed in the various tissues in the body. The resulting data are analyzed to provide quantitative spatial information of the in vivo biological process for which the tracer was designed. PET gives pharmaceutical research investigators the capability to assess biochemical changes or metabolic effects of a drug candidate in vivo for extended periods of time, and PET can be used to measure drug distribution, thus allowing the evaluation of the pharmacokinetics and pharmacodynamics of a particular drug candidate under study. Importantly, PET tracers can be designed and used to quantitate the presence of binding sites in tissues. Consequently, interest in PET tracers for drug development has been expanding based on the development of isotopically labeled biochemicals and appropriate detection devices to detect the radioactivity by external imaging.
Noninvasive nuclear imaging techniques such as PET have been particularly important in providing the ability to study neurological diseases and disorders, including stroke, Parkinson's disease, epilepsy, cerebral tumors and Alzheimer's disease Alzheimer's disease is the most common form of dementia. A PET radiotracer specific O-GlcNAcase would provide a powerful tool in demonstrating target engagement and pharmacodynamic activity and determining optimal doses in preclinical evaluation and clinical trials.
Disclosed herein are compounds that selectively inhibit the activity of O-GlcNAcase over the functionally related beta-hexosaminidases A and B, compositions that include the compounds, and methods of their use. Compounds disclosed herein as inhibitors of O-GlcNAcase possess both high potency and high permeability, and thus are useful in the treatment of diseases, disorders, or conditions that would benefit from the inhibition of O-GlcNAcase and reducing NFTs. The invention, also provides compounds which when radiolabeled are useful as PET radiotracers for imaging of O-GlcNAcase in vivo.